Purification Of RhBMP-2 By CHO Cells And Preliminary Study On The Effect Of New Polypeptide On Osteogenesis

Bone morphogenetic protein-2 (BMP-2) is one of at least 15 structurally and functionally related BMPs, which are members of the transforming growth factor P (TGF-P) superfamily. It is concerned in regulation of bone and cartilage formation and regeneration as well as embryogenesis and morphogenesis of various tissues and organs. The mature recombinant human BMP-2 is generated by the proteolytic removal of the signal peptide and propeptide.BMP-2 has been commercially available as an effective drug for a variety of clinical situations including long bone defect management, selected fracture and non-union repair, spinal fusion and maxillofacial disease treatment.BMPs have been isolated directly from demineralized bones, however with a complicated purification procedure and low yields. Alternatively, biologically active BMP-2 has been produced through in vitro refolding of inclusion bodies from Escherichia coli. However, a common disadvantage of these methods is the requirement of a complicated refolding procedure after several time-consuming steps of purification and concentration and the low overall yield is not satisfactory. As an alternative, BMP-2 has been obtained from mammalian cell cultures but with a low throughput purification method.The aim of the present work was to develop a convenient and feasible purification method with an appreciable yield and throughput for recombinant human BMP-2 production from Chinese hamster ovary cell line culture (CHO). Through analysis of the nature of BMP-2 and comparison of effects of different experimental conditions, we developed a convenient and low-cost purification method. The three-step procedure with a purification yield of 29% includes heparin affinity chromatography, Q-Sepharose ion exchange chromatography and Sephacryl S-200 gel filtration. About 0.26 mg of purified rhBMP-2 dimer with purity of more than 95% was obtained from 1 L of conditioned medium. It was shown to be biologically active by a histological analysis of ectopic bone formation. In conclusion, this method has the potential application for rhBMP-2 production from CHO.Osteoporosis is a skeletal disease characterized by the loss of bone mass and strength that leads to fractures. It is caused most often by an imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, which is the key means to understanding and treating osteoporosis. For treatment of osteoporosis, many drugs have been developed, including (1) bone resorption inhibitors, (2) bone formation accelerators, (3) bone mineralization drugs.Alkaline phosphatase (ALP) is a representative marker for osteoblastic differentiation. Bp08 is a new peptide synthesized in our laboratory. Using ALP activity as a marker, we investigated the role of bp08 in osteoblastic differentiation in MC3T3-E1 cells. We discovered that Bp08 dramatically increased ALP activity in MC3T3-E1 at the presence of BMP-2. Moreover, they could abolish the reducing effect of gw9662 on ALP activity. In addition, we tried to research the effect of Bp08 on bone metabolism with ovariectomized rat model of osteoporosis. The result revealed that Bp08 injected intravenously improved the bone mineral density of ovariectomized rats. Therefore, Bp08 might have potential application value in the treatment of orthopedic disorders.